The invention consists of a method to determine ethanol levels in all types of samples. This technique uses enzymes and chemicals to quantify or detect ethanol by visual means.
Chemical or enzymatic methods have been employed to determine ethanol content in biological fluids or breath after ingestion of ethanol. To do the analysis many of these methods require elaborate devices, a laboratory setting and special trained personnel. Described in previous patents (for example U.S. Pat. No. 3,223,488) some inventions permit an immediate visual approximation of blood ethanol from a breath ethanol sample which can be used by a chemical untrained person. The formerly patented devices used various chemicals such as potassium chromate to react with ethanol which resulted in a color change. The intensity of the color change or the time required for the ethanol sensitive chemical to change color was used to estimate the level of ethanol in the person's breath which in turn was used to estimate the blood ethanol. One severe disadvantage for these chemical methods is that they are not specific for ethanol and many substances present in biological samples but not in breath will interfere with the chemical test. Therefore these methods are useful to directly analyze breath samples but not other types of biological samples. The present invention describes a method that can directly analyze for ethanol in most biological samples because it is specific for ethanol and interfering substances are not present in sufficient levels in these samples to interact.
Some devices used to collect breath for the estimation of ethanol can be done by untrained personnel although it is complicated and cumbersome to use which can result in serious blood ethanol error approximations. The source of the error is that the device collects and analyzes all respiratory air while accurate blood ethanol estimates are based on alveolar ethanol levels. The invention in this disclosure describes a device that will permit quick blood ethanol estimation without this source of error.
An alternate method to chemical estimation of ethanol is the use of enzymes and chemicals. The technique has been used as a tool for analysis of many diverse chemicals. One enzyme that has been employed for the detection of ethanol is alcohol dehydrogenase (ADH). Patents describing various methods using ADH have been published (U.S. Pat. No. 3,941,659) but these methods required chemical trained individuals and laboratory equipment. The ADH method must be reduced to a simpler method before it is accessible to most individuals for ethanol analysis.
Alcohol oxidase, another enzyme has been reported to react with ethanol. This enzyme will react with substances in addition to ethanol however they are not in sufficient concentration in biological samples to interfere with ethanol analysis. In some biological samples this enzyme can be used for analysis of these chemicals.
The reaction between ethanol and alochol oxidase along with oxygen has produced hydrogen peroxide and acetaldehyde. The reaction that takes place is: ##STR1##
Three methods have used the above reaction to quantify ethanol. One method measured the formation of hydrogen peroxide while another method measured the amount of oxygen used in the above reaction and the third method measured the amount of acetaldehyde produced. Hydrogen peroxide can be quantified by using O-dianisidine, a colorless dye. This dye has been reported to react with hydrogen peroxide to form a colored oxidized product. The reaction was catalyzed by the enzyme peroxidase and was quantified by a spectrophotometer to determine the quantity of ethanol. The principle reaction underlying the invention in this disclosure is the measurement of hydrogen peroxide produced. This method and other methods that use alcohol oxidase to estimate ethanol levels are known but they are batch type operations that require fresh enzyme, laboratory instruments and chemical trained personnel. The device described in this disclosure has been reduced to a simple method that can be used by most individuals outside the laboratory setting using a stable alcohol oxidase enzyme. This aspect is the novelty of the device that is enhanced by a stable enzyme product. Previous to this report a method to stabilize alcohol oxidase at room temperature for months has not been available.
The availability of devices to estimate blood ethanol by visual methods use breath as the sample for ethanol analysis. A device is not presently available to directly detect or quantify ethanol in most biological samples without laboratory instruments. However, such devices would be extremely useful and is described in this disclosure. The detection or the ability to quantify ethanol in body fluids such as saliva, urine or blood would be useful to a individual, law enforcement officer or dispensor of alcoholic beverages to determine ethanol intoxication by a quick objective test. Emergency medical diagnosis and treatment could be improved if blood ethanol levels could be determined quickly.